The second messenger, cyclic GMP (cGMP) is believed to mediate the intracellular effects of nitric oxide and atrial natriuretic factor (ANF) on the relaxation of smooth muscle. This same smooth muscle relaxation is thought to be the mechanism of action of nitrovasodilators in mediating their therapeutic effects on angina pectoris. In this proposal, experiments are described to test the hypothesis that cGMP causes smooth muscle relaxation by activation of a specific cGMP-dependent protein kinase (cGK). Since multiple isoforms of cGK have been characterized previously, we will determine which of these isoforms are expressed in various types of smooth muscle using molecular and immunological techniques. Once the cGK isoforms relevant to smooth muscle relaxation have been identified, then these cGKs will be characterized biochemically to determine key properties of the enzymes relevant to their regulation of smooth muscle contraction. In vitro mutagenesis will be used to generate constitutively active and dominant negative forms of these cGKs for later in vivo analysis of the role that these enzymes play in regulation of smooth muscle relaxation. In addition, random peptide libraries will be screened in order to develop a specific pseudosubstrate inhibitor for the cGKs. These reagents will then be used to study the in vivo phosphorylation of substrate proteins in transfected smooth muscle cell lines as well as primary smooth muscle cell cultures. Two-dimensional gel electrophoresis will be used to characterize these substrates and to correlate their phosphorylation with regulation of smooth muscle cell functions such as regulation of intracellular calcium, densensitization of ANF receptors, and inhibition of mitogenesis. The dominant negative and constitutively active mutants as well as the pseudosubstate inhibitor will be used to definitively prove an obligatory role of cGK in relaxation of smooth muscle. Finally, important in vivo substrate proteins will be identified and cloned using a novel expression- based screening method. When completed, these studies will bridge a crucial gap in our knowledge concerning the mechanisms by which smooth muscle relaxation is regulated by ANF, nitric oxide, and nitrovasodilators.